Novel therapeutic agent that suppresses metastasis and proliferation of osteosarcoma and glioma

Abstract

Lysophosphatidic acid (LPA) has been found to function as a mediator that promotes invasiveness in osteosarcoma. In addition, it has been discovered that LPAR1, which is a receptor for LPA, is highly expressed in osteosarcoma and glioma, and that LPA-LPAR1 interaction participates in the metastasis and proliferation of osteosarcoma and glioma. When investigations were carried out based on these findings, it was discovered that an LPAR1 antagonist can provide a therapeutic agent that suppresses the metastasis and proliferation of osteosarcoma and glioma.

Description

A novel therapeutic agent that suppresses the metastasis and growth of osteosarcoma and glioma

[0001] The present invention relates to a novel therapeutic agent that suppresses the metastasis and growth of osteosarcoma and glioma, and in particular to a therapeutic agent for osteosarcoma and glioma that targets LPAR1.

[0002] Osteosarcoma is a malignant tumor that develops in bone and is histologically and genetically heterogeneous. While osteosarcoma is the most common malignant tumor occurring directly in bone, its incidence rate is low, at 1-3 per million, making it a rare cancer. However, because it is most commonly diagnosed in children and the AYA generation, and large doses of chemotherapy are administered before and after surgery, concerns are raised about the impact on growth and fertility, issues specific to the AYA generation, and the risk of developing new cancers in adulthood. Osteosarcoma is thought to first cause chromosomal instability through TP53 and RB1 gene mutations, followed by the development of polyclonal tumors with metastasis due to the occurrence of other oncogenic mutations (Non-Patent Documents 1 and 2). Therefore, its highly heterogeneous nature makes treatment difficult.

[0003] Osteosarcoma is not only a rare cancer, but also a highly heterogeneous tumor, as mentioned above. Therefore, the survival rate for metastatic osteosarcoma has not improved in the past 40 years. Lung metastasis is the most common, with 10-20% of patients presenting with pulmonary nodules at the time of initial diagnosis, and more than 80% of patients experiencing lung metastasis at the time of recurrence (Non-Patent Documents 3 and 4).

[0004] Although the survival rate of osteosarcoma patients has improved since the introduction of chemotherapy in the 1970s and the use of combined chemotherapy and surgical treatment, the survival rate of patients with metastasis is low, and there is no effective treatment for osteosarcoma patients with pulmonary metastasis, with the survival rate reportedly ranging from 19 to 37% (Non-Patent Documents 3, 5 to 8). Therefore, suppressing metastasis is an important issue in osteosarcoma treatment in order to improve prognosis.

[0005] Additionally, high-dose methotrexate, doxorubicin, cisplatin, and ifosofamide have been introduced as first-line chemotherapy treatments, but there is no consensus on the optimal combination therapy or treatments for metastasis and recurrent osteosarcoma. If therapeutic drugs targeting molecules specifically expressed in osteosarcoma could be obtained, it would be possible to provide new therapeutic drugs and treatments. Elucidating the molecules involved in the growth and metastasis of osteosarcoma is also important in establishing optimal treatments.

[0006] Gliomas are malignant brain tumors that arise from glial cells, with the most malignant type being called glioblastoma. In addition to headaches, various symptoms, such as paralysis of the limbs and impaired vision and vision, can occur depending on the location of the tumor and its growth. Gliomas are considered to be one of the tumors with a poor prognosis, as complete surgical removal is difficult when tumor cells infiltrate normal brain tissue. Furthermore, glioblastoma, the most malignant of all gliomas, has been reported to have causative gene mutations, such as IDH and p53 gene mutations, but no molecular targets that could lead to treatment have been reported.

[0007] Treatment for glioma involves surgery to remove as much of the tumor as possible while preserving motor and language functions. However, as mentioned above, it is difficult to remove the entire tumor, so radiation therapy and chemotherapy are used to prevent recurrence. Treatment involves administering temozolomide or bevacizumab in combination with radiation therapy, but recurrence is still common. Therefore, there is a need for the development of more effective therapeutic drugs, especially molecular targeted drugs.

[0008] Bousquet M, et al., 2016, Ann. Oncol. Vol. 27, pp.738-744.Ribi S, et al.,2015, Oncotarget, Vol. 6, pp.7727-7740.Kager L, et al., 2003, J.Clin. Oncol. Vol. 21, pp.2011-2018.Meyers PA, etal., 1993, J. Clin. Oncol. Vol.11, pp.449-453.AljubranAH et al., 2009, Ann. Oncol. Vol.20, pp.1136-1141.GokDurnali A, et al., 2016, PLoS One 2016; 11: e0152621.SalahS, et al. al., 2014, Mol. Clin. Oncol. Vol.2, pp.811-816.Briccoli A, 2010, Surg. Oncol. Vol.19, pp.193-199.O'Donnell VB et al., 2014, Circ.Res. Vol.114, pp. 1185-1203.Chatterjee M., 2020, J. Thromb.Haemost. Vol.18, pp. 543-557.

[0009] The present invention aims to provide effective therapeutic agents and methods for osteosarcoma and glioma. In particular, it aims to provide therapeutic agents that inhibit metastasis and tumor growth. Osteosarcoma metastasizes to the lungs at a relatively high rate, and there are no effective treatments for osteosarcoma patients with lung metastasis. Most patient deaths are due to respiratory failure caused by lung metastasis. Therefore, inhibiting metastasis, including lung metastasis, could improve prognosis. Furthermore, because gliomas are difficult to completely remove through surgical treatment, the development of therapeutic agents that inhibit tumor growth is anticipated. Effective molecular-targeted drugs have not yet been developed for osteosarcoma or glioma. Therefore, identifying molecules specifically expressed in these tumors and inhibiting their function could lead to the development of new therapeutic agents.

[0010] As will be described in detail below, the present inventors discovered that LPAR1 expression is high in osteosarcoma and completed the present invention. Furthermore, since LPAR1 expression is also high in glioma, the effects of LPAR1 antagonists were analyzed. This makes it possible to provide effective therapeutic agents for osteosarcoma and glioma, for which no effective therapeutic agents have been available until now.

[0011] The present invention relates to the following pharmaceutical compositions, prognostic diagnosis assisting methods, and drug selection methods: (1) A pharmaceutical composition for treating osteosarcoma or glioma, comprising as an active ingredient a compound that inhibits LPAR1 expression. (2) The pharmaceutical composition for treating osteosarcoma or glioma according to (1), wherein the compound that inhibits LPAR1 expression is an LPAR1 antagonist or a nucleic acid. (3) The pharmaceutical composition for treating osteosarcoma or glioma according to (2), wherein the LPAR1 antagonist is a low-molecular-weight compound. (4) The pharmaceutical composition for treating osteosarcoma or glioma according to (3), wherein the LPAR1 antagonist is ONO-7300243, BMS-986020, Ki16425, ONO-3080573, ONO-9780307, ​​ONO-9910539, Ki16198, AM095, AM966, SAR100842, BMS-986278, or an analog thereof. (5) A method for assisting prognosis in osteosarcoma patients, which comprises analyzing LPAR1 expression in diseased tissue obtained from the patient and determining that high LPAR1 expression indicates a high risk of metastasis. (6) A method for selecting a drug, which comprises analyzing LPAR1 expression in diseased tissue and determining that the patient should be administered an LPAR1 antagonist if LPAR1 expression is detected. (7) A method for screening for a pharmaceutical composition for treating osteosarcoma or glioma, comprising the steps of contacting a candidate substance with cells in which LPAR1 expression is observed and measuring changes in LPAR1 expression. (8) A method for treating osteosarcoma or glioma patients, comprising obtaining a specimen from the patient and analyzing LPAR1 expression, and if LPAR1 expression is observed, determining that the patient is a candidate for administration of a compound that inhibits LPAR1 expression, and administering the compound that inhibits LPAR1 expression.

[0012] These figures show that osteosarcoma cells have high platelet aggregation activity and that their invasive ability is enhanced by platelet releasates released from platelets. (A) shows the platelet aggregation activity of various osteosarcoma cells. (B) and (C) show that platelet releasates released from platelets enhance the invasive ability of osteosarcoma cells. (B) is a micrograph showing invaded cells, and (C) is a graph showing the number of invaded cells. These figures show that LPAR1 expression is upregulated in osteosarcoma. (A) shows the analysis of LPAR1 mRNA expression levels using RNA sequencing data from The Cancer Genome Atlas (TCGA) and TARGET. (B) shows the results of analyzing LPAR1 mRNA expression in osteosarcoma cell lines by qPCR, and (C) shows the results of analyzing LPAR1 protein expression by Western blotting. (D) shows the protein expression of LPAR1 in xenografts derived from osteosarcoma patients. This figure demonstrates that LPA released from activated platelets is important for the migration and invasion of osteosarcoma cells. (A) shows the release of LPA from platelets by adding MG-63 osteosarcoma cells to a platelet suspension, analyzed by ELISA. (B) shows the localization of phosphorylated AKT and F-actin in MG-63 osteosarcoma cells stimulated with LPA. (C) shows the effect of LPA on cell migration in various osteosarcoma cells and the cancellation of this effect by the LPAR antagonist Ki16425. (D) shows the effect of platelet release on the invasion of osteosarcoma cells and the cancellation of the effect of platelet release by Ki16425. (E) shows the effect of Ki16425 on osteosarcoma cell proliferation. Figures showing that LPA-LPAR1 interaction is important for osteosarcoma cell invasion. (A) Figure showing the establishment of LPAR1 knockout cells using MG-63 osteosarcoma cells. (B) and (C) Figures showing the effect of LPA on the invasive ability of LPAR1 knockout cells. (D) and (E) Figures showing the effect of platelet releasates on the invasive ability of LPAR1 knockout cells. Figures showing that LPAR1 plays an important role in lung metastasis of osteosarcoma.(A) shows the luminescence of Akaluc luciferase-transfected MG-63 osteosarcoma cells (MG-63 / Akaluc / sgCTRL) and LPAR1-knockout MG-63 osteosarcoma cells (MG-63 / Akaluc / sgLPAR#1), and (B) shows cell proliferation ability. (C) and (D) show the results of in vivo imaging analysis of MG-63 / Akaluc / sgCTRL and MG-63 / Akaluc / sgLPAR#1 cells intravenously injected, followed by administration of AkaLumine on day 0 (the day of injection) and day 7. (A) shows the suppression of lung metastasis of osteosarcoma by an LPAR1 antagonist. (B) shows the experimental procedure. (B) and (C) are in vivo imaging analysis results showing that administration of the LPAR1 antagonist ONO-7300243 followed by injection of HuO9 / Akaluc osteosarcoma cells suppresses lung metastasis. Figures showing that LPAR1 is involved in osteosarcoma cell proliferation. (A) is a diagram showing that cell proliferation is suppressed in clonal lines obtained by knocking out LPAR1 in MG-63 osteosarcoma cells and G-292 clone A141B1 (G-292) cells. (B) is a diagram showing that apoptosis is induced by knocking down LPAR1 with siRNA in MG-63 and HuO9 osteosarcoma cells. Figures showing that administration of an LPAR1 antagonist exerts an antitumor effect in an osteosarcoma xenograft model. (A) shows the LPAR1 administration schedule, and (B) shows the change in tumor volume over time. (B) A diagram showing that LPAR1 antagonist administration exerts an antitumor effect in a glioblastoma xenograft model. (A) The LPAR1 antagonist administration schedule is shown, and (B) the change in relative tumor volume over time is shown.

[0013] The present inventors discovered that osteosarcoma cells have high platelet aggregation activity and that platelet releasates released from activated platelets enhance the invasive ability of osteosarcoma cells. Further analysis revealed that lysophosphatidic acid (LPA) released from activated platelets functions as a mediator that enhances the invasive ability of osteosarcoma cells. Furthermore, they found that the expression of LPA receptor 1 (LPAR1) was significantly increased in osteosarcoma cells and patient-derived xenografts, leading them to conclude that LPA-LPAR1 interaction is involved in the distant metastasis of osteosarcoma.

[0014] Furthermore, using a mouse model, we found that administration of an LPAR1 antagonist, specifically ONO-7300243, inhibited lung metastasis, and administration of BMS986020 inhibited tumor growth. Furthermore, we found that cells in which LPAR1 expression was knocked out or knocked down exhibited inhibited proliferation and induced apoptosis, indicating that osteosarcoma growth can be inhibited by inhibiting LPAR1 expression. Furthermore, since LPAR1 is also highly expressed in gliomas, we analyzed the effects of an LPAR1 antagonist and found that it exerted an antitumor effect.

[0015] As described above, LPA-LPAR1 interaction is involved in the metastasis and proliferation of osteosarcoma and glioma, and therefore, any LPAR1 antagonist that suppresses metastasis and proliferation may be used. Here, a low molecular weight compound is used, but such compounds include, in addition to the ONO-7300243, BMS-986020, and Ki16425 used here, for example, ONO-3080573, ONO-9780307, ​​ONO-9910539, Ki16198, AM095, AM966, SAR100842, BMS-986278, and analogs thereof. Furthermore, antibodies or polypeptides that bind to LPAR1 and inhibit its function may also be used. Furthermore, LPAR1 expression itself may be suppressed by nucleic acids such as siRNA, antisense RNA, shRNA, and miRNA.

[0016] In addition to existing LPAR1 antagonists, compounds that suppress LPAR1 expression may also be screened for and used. Compounds that suppress LPAR1 expression may be screened by adding a candidate substance to a culture medium of cells expressing LPAR1, specifically osteosarcoma cells or glioma cells, and using a decrease in LPAR1 expression as an index.

[0017] Furthermore, in the case of osteosarcoma patients, since LPAR1 expression is closely related to bone metastasis, measuring LPAR1 expression in tumor tissue makes it possible to determine the risk of distant metastasis, i.e., prognosis. Specifically, LPAR1 expression is detected in osteosarcoma tissue obtained by surgery or biopsy, and if LPAR1 expression is high, it can be determined that there is a high possibility of distant metastasis. If there is a high possibility of distant metastasis, distant metastasis can be prevented by taking preventive measures such as administering an LPAR1 antagonist. Furthermore, since LPAR1 inhibitors are thought to be effective when LPAR1 expression is high not only in osteosarcoma and glioma but also in other cancer types, testing LPAR1 expression in patient samples can determine which patients should be treated with an LPAR1 inhibitor.

[0018] The following data are presented to explain the findings. While platelet-cancer interactions and the release of bioactive molecules from activated platelets have been reported to be important in hematogenous metastasis of epithelial tumors, their role in sarcoma remains unclear. To clarify whether osteosarcoma cells interact with and activate platelets, platelet aggregation assays were performed using eight osteosarcoma cell lines (Figure 1(A)). Human osteosarcoma cells were obtained from the ATCC, the RIKEN BioResource Research Center, and the JCRB Cell Bank and cultured according to their respective recommended protocols.

[0019] Platelets were isolated by a conventional method from blood obtained from healthy individuals who had not taken antiplatelet drugs for at least 10 days prior to blood collection. ８ / mL of modified Tyrode's buffer (137 mM NaCl, 11.9 mM NaHCO ３ , 0.4 mM Na ２ HPO ４, 2.7mM KCl, 1.1mM MgCl ２ , 5.6 mM glucose) and 1.2 mM CaCl ２ The platelet suspension was added with 200 μL of the cell suspension (5 × 10 ６ 10 μL of a cell suspension (100 μL / mL) or PBS was added, and analysis was carried out at 37°C for 30 to 60 minutes. Platelet aggregation was measured using an aggregometer (MCM HEMA Tracer 313M, SSR Engineering) (Figure 1(A)).

[0020] Compared with the lung adenocarcinoma cell line A549 used as a negative control, all osteosarcoma cells had high platelet activation ability. Collagen (10 μg / mL, added 10 μL) was used as a positive control. These results demonstrated that all osteosarcoma cells used in the analysis had high platelet aggregation ability, i.e., platelet activation ability.

[0021] We investigated the possibility that bioactive molecules released from activated platelets affect the invasive ability of osteosarcoma cells. The platelet aggregation assay reaction mixture was collected and treated with 0.5 μM prostaglandin I. ２ The supernatant was used as a platelet release product containing bioactive molecules released from activated platelets.

[0022] Osteosarcoma cells MG-63, HuO9, and G-292 were placed in the insert (upper chamber) of a Matrigel invasion chamber (Corning) at 1.5 × 10 ５ The cells were seeded at 0.5 mL per 1000 cells, and the platelet releasate was placed in the lower chamber. After incubation at 37°C for 22-24 hours, the cells on the upper surface of the insert were completely wiped and fixed with 4% paraformaldehyde. The cells on the lower surface of the insert membrane, i.e., the invading cells, were stained with 1% crystal violet (Figure 1(B)). The number of invading cells was counted, and the relative cell ratios are shown in Figure 1(C). In both osteosarcoma cell lines, the addition of platelet releasate significantly increased the number of invaded cells.

[0023] It is known that not only peptides but also lipids function as mediators in platelet release. To analyze whether peptides or lipids mediators are involved in the invasive ability of osteosarcoma cells, we performed heat treatment at 95°C for 10 minutes, a condition that denatures most proteins, and analyzed the invasive ability in a Matrigel invasion chamber (Fig. 1(B) and (C)). As a result, the effect was not abolished by heat treatment, suggesting that the mediator is lipid.

[0024] Mass spectrometry analysis has reported that activated platelets release lipid mediators such as TxA2, S1P, and LPA (Non-Patent Documents 9, 10). Although data are not presented here, the expression of these lipid mediator receptors (TBA2R, S1PR1-5, LPAR1-6) in various tumors was examined using RNA sequencing data from the TCGA and TARGET databases. High expression of LPAR1, LPAR6, S1PR1, and S1PR3 was observed in osteosarcoma. Furthermore, when comparing various cancer types, LPAR1 was found to be most highly expressed in osteosarcoma (OS) and sarcoma (SARC) (Figure 2(A)). Because the gene expression data from TCGA and TARGET are tumor tissue data, they also include data on stromal cells, epithelial cells, immune cells, and other cells in addition to tumor cells. Therefore, although the data are not shown here, we investigated using the Cancer Cell Line Encyclopedia (CCLE) database, which reflects gene expression in cancer cell lines. We found that LPAR1 expression in several osteosarcoma cell lines was significantly higher than that in Ewing sarcoma family tumors and chondrosarcoma.

[0025] Furthermore, we confirmed by qPCR (Figure 2(B)) and Western blotting (Figure 2(C)) that LPAR1 was highly expressed in six of the eight osteosarcoma cell lines: MG-63, HuO9, HuO-3N1, G-292, NY, and SJSA-1. Western blotting also revealed that approximately 80% (15 / 19) of osteosarcoma-derived xenograft samples established from patients were LPAR1-positive (Figure 2(D)).

[0026] Whether LPA is released from platelets upon interaction with osteosarcoma cells was analyzed by ELISA (human lysophosphatidic acid ELISA kit, Cusabio). 200 μL of platelet suspension and 5 × 10 ４ Each MG-63 cell or MG-63 cells alone was incubated at 37°C for 30 minutes. ２ After centrifugation, LPA was detected by ELISA. As shown in Figure 3(A), no LPA was detected in the supernatant of MG-63 cells alone, whereas a large amount of LPA was detected when MG-63 cells were mixed with platelets. Furthermore, significant LPA release was observed compared with the supernatant of non-activated platelets (control).

[0027] LPA receptors are G protein-coupled receptors, and LPAR1 is a G αｉ／０ , G αｑ／１１ , Gα １２／１３ LPA activates three G proteins, which are known to activate signal transduction pathways, including the PI3K / AKT pathway. MG-63 cells were cultured overnight in serum-free MEM medium and then treated with 100 nM LPA for 4 hours. Immunostaining was performed using standard methods with an anti-phosphorylated AKT antibody (an antibody that detects phosphorylation of S473), rhodamine-labeled phalloidin (which binds to F-actin), and Hoechst 33342 (which stains nuclei), followed by microscopic observation (Figure 3(B)). In LPA-treated cells, activated AKT was observed, and as indicated by the arrow, it was also localized in the same region as F-actin. Although the results are not shown here, we performed time-lapse microscopic analysis to analyze the effect of LPA on osteosarcoma cells. LPA treatment induced the formation of numerous pseudopodia (protrusions). Furthermore, not only in MG-63 cells but also in HuO9 cells, co-localization of phosphorylated AKT and F-actin was observed at the protrusions of the cell membrane.

[0028] Next, the effects of LPA on migration and invasion were analyzed (Figure 3(C)). Migration was measured using a transwell chamber (Corning, 8.0 μm pore). 1 × 10 cells suspended in 0.3 mL of serum-free MEM medium were placed in the insert. ５ MG-63, HuO9, or G-292 cells were seeded in 1.25 mL of serum-free MEM medium containing 10 nM LPA as a chemoattractant in the lower chamber. After 4-6 hours of incubation, the cells were fixed and analyzed for migration (LPA). Cells were also pretreated with 100 nM Ki16425 (Santa Cruz Biotechnology) for 1 hour, harvested, seeded on inserts, and analyzed for migration in the presence of 100 nM Ki16425 (LPA+Ki16425). While LPA significantly enhanced migration in all cell types, Ki16425-treated cells significantly inhibited migration even with the addition of LPA.

[0029] Invasion ability was also analyzed using a Matrigel invasion chamber, as shown in Figure 1(C) (Figure 3(D)). Analysis of platelet release products after adding them to the lower chamber revealed that the platelet release products induced cell invasiveness in all cell types. Furthermore, prior treatment of cells with Ki16425 significantly inhibited invasive ability. As shown in Figure 3(E), when G-292 and MG-63 cells were cultured with Ki16425 for 72 hours, cell viability decreased at concentrations of 500 nM or higher. Migration and invasion tests were performed using relatively low concentrations of 100 nM for 4-6 hours (migration) or 22-24 hours (invasion), and no cytotoxicity was observed under these conditions. Therefore, it was confirmed that the inhibition of migration and invasion by Ki16425 was not due to cytotoxicity. These results indicate that the biologically active molecule in platelet release is LPA, and that LPA mediates the enhancement of the migration and invasion abilities of osteosarcoma cells.

[0030] Ki16425 has an inhibitory effect on LPAR1, LPAR2, and LPAR3, although the degree of inhibition varies. To determine which LPAR functions in osteosarcoma invasiveness, LPAR1 knockout cells were established from MG-63 cells using the CRISPR / CAS9 system. The established cells are designated sgLPAR1#1-3. Western blotting confirmed that LPAR1 expression was lost in all sgLPAR1#1-3 (Figure 4(A)).

[0031] These knockout cells were used to analyze their invasive ability. Each cell was placed in an insert at 1 × 10 ５ 10 nM LPA was added to the lower chamber of the Matrigel invasion chamber, and after 22-24 hours, the cells were fixed, stained with crystal violet, and counted (Figures 4(B) and (C)). Both knockout cells showed significantly reduced invasive ability compared to the control.

[0032] Similar analysis was performed on platelet releasates released from activated platelets. Platelet releasates equivalent to 10 nM LPA were added to the lower chamber and cultured for 22-24 hours, after which the cells were fixed, stained, and analyzed (Figures 4(D) and (E)). Neither knockout cell line showed enhanced invasive potential due to platelet releasates. These results suggest that lung metastasis of osteosarcoma is mediated by LPA-LPAR1 interaction.

[0033] Next, we analyzed the function of LPAR1 in lung metastasis of osteosarcoma in vivo using a mouse model. Akaluc luciferase was introduced into MG-63 / sgCTRL and MG-63 / sgLPAR#1 cells to obtain Akaluc luciferase-expressing cells, i.e., MG-63 / Akaluc / sgCTRL and MG-63 / Akaluc / sgLPAR#1 cells. Various cell numbers, as shown in Figure 5(A), were seeded and treated with 50 μM AkaLumine-HCl. Luminescence was measured using a Mithras LB940 plate reader (Berthold Technology). The resulting cells were confirmed to emit light with comparable intensity depending on the cell number. Cell proliferation was also measured using CellTiter-Gro reagent (Promega). No significant difference in proliferation was observed between the two types of cells (Fig. 5(B)).

[0034] Female SCID-beige mice (C.B-lgh-1b / GbmsTac-Prkdc ｓｃｉｄ -Lyst ｂｇ N7, Charles River Japan Co., Ltd.) ６ MG-63 / Akaluc / sgCTRL or MG-63 / Akaluc / sgLPAR#1 were injected intravenously, and 3 hours and 7 days later, 100 μL of 5 mM AkaLumine-HCl was injected intraperitoneally, and in vivo bioluminescent imaging (BLI) was performed. BLI was performed using an IVIS imaging system (PerkinElmer). Figure 5(C) shows the image, and (D) shows the total flux measured by IVIS. On the day of intravenous cell injection (day 0), both types of cells were trapped in the lungs to a similar extent. However, after 7 days (day 7), MG-63 / Akaluc / sgCTRL cells had engrafted in the lung, whereas LPAR1 knockout cells, MG-63 / Akaluc / sgLPAR#1, had not. A significant difference was observed in total flux, indicating that LPAR1 plays an important role in lung metastasis.

[0035] Next, the effect of an LPAR1 antagonist was examined. Analysis was performed in a mouse model using ONO-7300243 (Cayman Chemical), an LPAR1 inhibitor. As shown in Figure 6(A), after oral administration of ONO-7300243, 1 × 10 ６ HuO9 / Akaluc cells were injected intravenously and BLI was performed in the same manner. The experiment was carried out using ONO-7300243 at different doses of 10 mg / kg and 30 mg / kg.

[0036] BLI was performed 1.5 to 3 hours after administration, and 1 and 2 days later (Figure 6(B)). Immediately after administration, HuO9 / Akaluc cells were observed to be trapped in the lungs at all doses. With the passage of time (1 and 2 days), it was confirmed that the number of osteosarcoma cells trapped in the lungs decreased in the ONO-7300243-treated group compared to the vehicle-treated group.

[0037] The total flux measured by IVIS (Figure 6(C)) showed no significant difference among the groups on the day of cell injection, but a significant difference was observed between the vehicle-administered group and the 30 mg / kg-administered group one day later, and a significant difference was also observed between the 10 mg / kg-administered group and the 30 mg / kg-administered group two days later, demonstrating a dose-dependent decrease in osteosarcoma cells trapped in the lungs.

[0038] As shown above, LPAR1 knockout and treatment with an LPAR1 antagonist suppressed metastasis. Next, we analyzed the effect of LPAR1 expression on cell proliferation. As shown in Figure 3(E), experiments using an LPAR1 antagonist did not show any significant effect on cell proliferation. Therefore, we used a more sensitive system to analyze whether LPAR1 expression affects cell proliferation.

[0039] Using the CRISPR / Cas9 system, LPAR1 was knocked out in osteosarcoma cells MG-63 and G-292, and then clones of each cell were obtained and compared for cell proliferation ability (Figure 7(A)). In both cell lines, suppression of cell proliferation was observed in the LPAR1 knockout cell lines.

[0040] We investigated the mechanism by which cell proliferation is suppressed. LPAR1 was knocked down in MG-63 and HuO9 cells using siRNA (Dharmacon) targeting LPAR. Knockdown of LPAR1 was confirmed to increase cleaved PARP (Cl-PARP), an apoptosis marker, suggesting that LPAR1 knockdown induces apoptosis. Therefore, it is believed that not only LPAR1 antagonists but also siRNA-mediated suppression of LPAR1 expression induces apoptosis in osteosarcoma cells and exerts an antitumor effect.

[0041] These results suggest that LPAR1 antagonists may induce apoptosis and suppress osteosarcoma cell growth. Therefore, we investigated whether administration of an LPAR1 antagonist suppresses the growth of osteosarcoma cells subcutaneously transplanted into mice (Figure 8).

[0042] Female SCID-beige mice were subcutaneously inoculated with 8.2 × 10 osteosarcoma cells G-292. ５ Cells were transplanted (Day 0), and starting the day after transplantation (Day 1), the LPAR1 antagonist BMS-986020 was orally administered at 30 mg / kg for 5 consecutive days followed by 2 days off (Figure 8(A)). Tumor growth was assessed by measuring the long and short diameters of the tumor and calculating the long diameter (mm) x [short diameter (mm)]. ２ The tumor volume was calculated as 1 / 2 of the normalized value and analyzed.

[0043] Tumor volume was measured from day 14 after tumor implantation, and a significant tumor growth inhibitory effect was observed in the LPAR1 antagonist-administered group from day 14 onward ( Fig. 8(B) ). Since the LPAR1 antagonist was observed to have an antitumor effect, it can be expected to not only inhibit lung metastasis of osteosarcoma but also lead to remission.

[0044] As shown in Figure 2 (A), LPAR1 is highly expressed in glioblastoma (GBM) and glioma (LGG), following osteosarcoma and sarcoma. Therefore, LPAR1 antagonists are expected to be effective in suppressing metastasis and cell proliferation in glioma as well. As mentioned above, glioblastoma is the most malignant tumor among gliomas, so the migration ability of glioblastoma cell lines was analyzed by adding LPA. An analysis was conducted to determine whether the motility of glioblastoma cells is enhanced in the presence of LPA. Human glioblastoma cells Onda7 (obtained from JCRB Cell Bank), YKG-1 (obtained from JCRB Cell Bank), and 42-MG-BA (obtained from DSMZ) were each cultured at 1 x 10 ５ Cells were seeded into a transwell chamber, and 100 nM LPA was added to the lower chamber as a migration factor. After 24 hours, cells that had migrated to the lower chamber were stained with crystal violet and quantified. Furthermore, before harvesting, cells were pretreated with 100 nM Ki16425, an LPAR antagonist, for 1 hour, and then their migration was analyzed in the presence of 100 nM Ki16425. While LPA significantly enhanced the migration of all cells, cells pretreated with Ki16425 significantly inhibited the migration of cells, even after the addition of LPA (Figure 9).

[0045] Next, we investigated whether LPAR1 antagonists have an effect on the viability of glioblastoma cells. Human glioblastoma cells, Onda7, YKG-1, and 42-MG-BA, were seeded in 96-well plates at 1,500 to 3,000 cells and treated with varying concentrations of the LPAR1 antagonists Ki16425, BMS-986020, and ONO-7300243. Cell viability after 72 hours was measured using CellTiter-Gro reagent (Figure 10(A)). A decrease in cell viability was observed for all cells and all LPAR1 antagonists.

[0046] Next, we analyzed the effect of LPAR1 knockdown on glioblastoma cell viability (Figure 10(B)). Onda7 and YKG-1 cells were seeded and treated with two types of siRNA (Dharmacon) targeting LPAR1. Cell viability was measured 4 days later using CellTiter-Gro reagent. When LPAR1 expression was inhibited by siRNA, a significant decrease in viability was observed in both cells. Furthermore, an increase in cleaved PARP (Cl-PARP), an apoptosis marker, was observed, suggesting that apoptosis was induced by LPAR1 knockdown. These results suggest that reducing LPAR1 expression in glioblastoma can be expected to have antimetastatic and antiproliferative effects. In other words, LPAR1 antagonists can be used as molecularly targeted drugs for treatment.

[0047] We also investigated whether LPAR1 antagonists have an antitumor effect on glioblastoma. ５ The tumors were intracranially transplanted into female SCID-beige mice. Starting three weeks after transplantation, the LPAR1 antagonist BMS-986020 was orally administered at 50 mg / kg or 100 mg / kg on a five-day administration schedule followed by two days off (Figure 11(A)). Tumor volume was determined by quantifying the enzyme activity of AkaLuc luciferase stably expressed in Onda7 cells using an IVIS imaging system (Figure 11(B)).

[0048] In the BMS-986020 group, even at a dose of 50 mg / kg, inhibition of glioblastoma cell growth was observed starting 2 weeks after the start of administration (5 weeks after transplantation), and the relative tumor volume was almost equivalent to that at the time of transplantation. From 7 weeks after transplantation onwards, a significant difference was observed compared to the control. It was observed that LPAR1 antagonists also exhibit antitumor effects against glioblastoma.

[0049] As described above, the inventors have demonstrated that LPAR1 is significantly involved in the mechanism of osteosarcoma metastasis. Furthermore, based on the elucidation of the mechanism of metastasis, they demonstrated in a mouse model that administration of an LPAR1 antagonist can inhibit metastasis. As described above, osteosarcoma metastasizes to the lungs at a high rate, and there is no effective treatment for osteosarcoma patients with lung metastasis. Most patients die from respiratory failure due to lung metastasis. Considering this, the ability of LPAR1 antagonists to inhibit lung metastasis of osteosarcoma is highly useful for providing a new treatment for osteosarcoma and improving prognosis. Furthermore, they have shown that inhibition of LPAR1 expression inhibits osteosarcoma cell proliferation and induces apoptosis. In other words, administration of an LPAR1 antagonist inhibits not only metastasis but also osteosarcoma growth. Treatment of osteosarcoma by inhibiting LPA-LPAR1 interaction is believed to be a novel therapy capable of inhibiting osteosarcoma metastasis and growth.

[0050] Furthermore, LPAR1 antagonists have been shown to be effective not only for osteosarcoma but also for glioma, which also has high LPAR1 expression. Gliomas are difficult to treat surgically depending on the site of development, and are intractable tumors in which it is difficult to completely remove tumor cells. LPAR1 antagonists are thought to be a new treatment option for gliomas as well.

Claims

1. A pharmaceutical composition for treating osteosarcoma, comprising a compound that suppresses LPAR1 expression as an active ingredient.

2. The compound that suppresses LPAR1 expression is an LPAR1 antagonist or a nucleic acid. The pharmaceutical composition for treating osteosarcoma according to Claim 1, characterized in that.

3. The pharmaceutical composition for treating osteosarcoma according to Claim 2, characterized in that the LPAR1 antagonist is a low molecular weight compound.

4. The LPAR1 antagonist is ONO-7300243, BMS-986020, Ki16425, ONO-3080573, ONO-9780307, ONO-9910539, Ki16198, AM095, AM966, SAR100842, BMS-986278, and analogs thereof. The pharmaceutical composition for treating osteosarcoma according to Claim 3, characterized in that.

5. A method for assisting in the prognostic diagnosis of an osteosarcoma patient, comprising analyzing the LPAR1 expression in diseased tissue obtained from the patient, and a method for assisting in prognostic diagnosis, wherein when the LPAR1 expression is high, the risk of metastasis is considered high.

6. Analyze the LPAR1 expression in diseased tissue in an osteosarcoma patient. A method for selecting a medicament, wherein when LPAR1 expression is detected, it is determined that the patient is a candidate for administration of the LPAR1 antagonist according to Claim 2.

7. A screening method for a pharmaceutical composition for treating osteosarcoma, comprising a step of contacting a candidate substance with cells in which LPAR1 expression is observed, and a screening method characterized by including a step of measuring a change in LPAR1 expression.